Antibodies have been immobilized on the surfaces of various inorganic solid materials for producing immunosensor, protein chip, diagnostic kit, etc. The technique to regulate orientation in order to expose the antigen binding site of an antibody on the surface without damaging the activity and binding capacity peculiar to the antibody and thus to immobilize the antibody on the various solid substrates as a form of monolayer is very important since it is directly involved in the detecting sensitivity of a sensor or a chip.
The conventional methods to immobilize an antibody on the surface of a solid substrate rely on physical adsorption or covalent bond formation of a protein. However, the immobilization of an antibody by the conventional methods has disadvantages of protein denaturation, random orientation, decreased binding capacity of the immobilized antibody to the target antigen resulting from random chemical modifications.
To overcome the above problems, antibody immobilization techniques have been developed by using microorganism-originated antibody binding proteins (protein A, protein G, protein A/G or protein L) binding specifically to the corresponding antibody. These proteins bind strongly to a specific region of an antibody that is not involved in the antigen-antibody reaction, immobilizing the antibody on a solid substrate to allow the approach of an antigen. The interaction between the above proteins and antibodies does not require any chemical modification process, suggesting that the unique antibody functions are not damaged. However, it is very difficult to regulate orientation of the proteins during immobilization on a solid substrate, and as a result, the antibody immobilization efficiency might be reduced. Recent studies have been focused on the modification of an antibody binding protein by genetic engineering and chemical approaches to overcome the above problem.
The antibody immobilization using an antibody binding protein has many advantages, compared with the method based on physical adsorption, but still has a problem of protein denaturation by many environmental factors including physical and chemical factors, suggesting that long-term storage is difficult. It is also almost impossible to treat a specific target region only in case a chemical modification is necessary in a specific region of a protein. It is an urgent request, to overcome such problems, to develop a novel method for antibody immobilization using a low molecular weight material having high stability and facilitating immobilization on a solid substrate. Antibody immobilization methods have been developed using a dendrimer, iron ion or calixcrown derivatives, but these methods do not have the control of orientation and selectivity, suggesting that the antibody does not have protein specificity, that is it can be bound to almost every proteins with similar binding capacity which is though not very strong.
To screen a novel low molecular substance for protein immobilization in order to overcome the disadvantages of the conventional antibody binding protein or low molecular substance for antibody immobilization, the present inventors carried out wide document analysis to investigate which low molecular substance could be used as an adsorption material for antibody separation and as a therapeutic agent based on antibody binding. The present inventors selected three kinds of peptides binding selectively to IgG as candidates for the low molecular substance of the invention (DeLano W L et al., Science 287:1279-1283, 2000; Yang H et al., J Peptide Res 66(Suppl. 1):120-137, 2006; Fassina G et al., J Mol Recognit 11:128-133, 1998), and prepared peptide hybrids for protein immobilization by modifying chemical structures of the peptides, and finally completed this invention by confirming that the peptide hybrids for protein immobilization had binding capacity about antibody and antigen on the solid substrate for the immunosensor and antibody chip.